: Synaptic loss and neurodegeneration in the neocortex and limbic system in patients with Alzheimer's disease (AD) results in characteristic cognitive alterations. Therefore, to better understand the pathophysiology of AD it is necessary to understand the cellular and molecular mechanisms involved in synaptic damage. During the course of funding for this project the applicants have shown that abnormal functioning of amyloid precursor protein (APP) at the synaptic site might be involved in mechanisms of neurodegeneration in AD. For the renewal period, they hypothesize that abnormal APP functioning might result in deficient functioning of glutamate transporters (GT) which in turn results in increased levels of glutamate at the synaptic cleft and exitotoxicity/neurogeneration. Furthermore, they hypothesize that low density lipoprotein receptor-related protein (LRP) plays an important role in mediating the effects of APP on the GT. Therefore, the main objectives of this revised version of the project are to 1) characterize in vivo the specifity of interactions between APP and GT; 2) assess the in vivo role of putative receptors of APP in mediating the effects of APP on GTs; 3) determine the subcellular mechanisms mediating the effects of APP on GT; 4) determine if altered GT activity, promoted by APP, plays a role in neuroprotection and neurodegeneration. To achieve these aims, levels of mRNA and protein expression and activity (Kd, Bmax) for neuronal and glial GTs will be assessed in the nervous system of challenged (kainic acid injection) and naive NSE-APP transgenic, APP- and LRP-deficient mice by RNAse protection assay, immunochemistry, and competitive and saturated uptake binding assays with D-and L- aspartate. Neuronal and synaptic integrity will be evaluated by immunoquantification of synaptophysin (SYN, synaptic marker) and microtubule associated protein 2 (MAP2, dendritic marker). Additional studies of the role of APP effect on GTs will be performed in a human neuronal cell line (HNT) after treatment with APP, LRP blockers, protein kinase C (PKC) inhibitors and stimulators, and calcium channel blockers. They hypothesize that APP binding to LRP triggers, in addition to the internalization of the ligand, the activation of PKC, which in turn results in GT activity regulation. In this context, they propose to study the role of APP and LRP interactions in regulating the levels of excitotoxins at the synaptic site and to determine how dysfunction of this process might result in neurodegeneration. These studies will help to better understand the mechanisms of synaptic pathology in AD and to develop potential neuroprotective agents that will operate by enhancing the GT activity.